Arrangement for optimizing the pulse shape in a laser scanning microscope

ABSTRACT

A Device for coupling a short pulse laser into a microscope beam path, wherein the spectral components of the laser radiation are spatially separated by means of a dispersive element, the individual spectral components are manipulated and are then spatially superimposed again by means of another dispersive element.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.10/916,813, the entire disclosure of which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

a) Filed of Invention

At present, nonlinear contrasts such as two-photon absorption or secondharmonic generation (SHG) are used to an increasing extent inmicroscopy, e.g., for examination of biological preparations. It isadvantageous to use short pulse lasers to provide the energy needed toexcite nonlinear effects. In this connection, the peak pulse powershould be as high as possible and the pulse length at the location ofthe specimen should accordingly be as small as possible to preventdamage to the preparation simultaneously. Short pulse lasers supplylight pulses, for example, of several 10 fs at a repetition rate ofseveral 10 MHz. Accordingly, they have the advantage that they emitextremely high peak pulse energies accompanied at the same time by lowaverage output.

It is disadvantageous that the short pulses on the path through themicroscope to the specimen change due to the group velocity dispersion(GVD)—usually, they become longer.

b) Description of the Related Art

In order to compensate for pulse lengthening, corresponding changes(prechirp devices) have been suggested (DE 19622353). Further, adaptiveoptics have been provided in DE 19733193. The described devices aresuitable for compensation of second-order dispersion.

However, higher-order dispersions which cannot be determined beforehandmust be taken into account, e.g., in biological preparations. Further,higher-order dispersions occur in the optical components in amicroscope. Therefore, it is not possible to create optimum conditionsfor the excitation of nonlinear contrasts by conventional techniques.

In conventional fluorescence microscopy, different dyes are used forspecific tagging of biological preparations. These dyes are subsequentlyexcited by different light wavelengths. In preparations of this kind,simultaneous excitation of the various dyes is usually carried out usingmultiphoton excitation. On the one hand, this is advantageous becauseonly one light wavelength is needed for excitation. On the other hand,it is disadvantageous when the emission wavelength bands of theindividual dyes overlap because the dyes can then no longer bespectrally separated.

OBJECT AND SUMMARY OF THE INVENTION

It is the primary object of the invention to overcome the abovedescribed disadvantages.

In accordance with the invention, a device for coupling a short laserinto a microscope beam comprises a dispersive element for spatiallyseparating the spectral components of the laser radiation, means formanipulating individual spectral components and another dispersiveelement for spatially superimposing the manipulated individual spectralcomponents.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a block diagram of the arrangement in accordance with theinvention;

FIG. 2 a is a schematic representation of a 4f system;

FIG. 2 b is a schematic representation of a folded 4f system; and

FIG. 3 shows schematically the dispersive splitting and continuation ofa red component r and a blue component b passing the manipulator and thewavelength shape along a direction x to the manipulator.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The light pulses proceed from the short pulse laser KL to the pulseshaper PF. The latter is shown schematically in FIG. 2 a. In the pulseshaper PF, the incident beam (beam in) is spatially split into thespectral components of the light pulses in a first dispersive element(1) comprising, e.g., a grating or prisms. A Fourier plane is thengenerated by means of an achromatically corrected lens or lens group L1(FIG. 2).

This plane (focal plane) is characterized in that the individualspectral components of the light pulses are spatially separated.Considered mathematically, the transformation into this planecorresponds to a Fourier transform. In this plane, a spatial lightmodulator (2) (SLM) is used in transmission. The modulator is alsoreferred to herein as a manipulator of spectral components. Generally,it comprises a matrix of nematic liquid crystals (e.g., SLM-S160/h,Jenoptik LOS) in helical or parallel arrangement. The transmission andphase displacement of the corresponding spectral components can beadjusted by a corresponding electronic arrangement of the individualpoints of the matrix. The spatial separation of the spectral componentsof the light pulses is then canceled by a second identical lens L2 and asecond dispersive element (3) (beam out) identical to the firstdispersive element. This process corresponds to the inverse transform inthe time domain. Therefore, the time behavior of the light pulses can becontrolled by means of phase modulation or amplitude modulation. Thearrangement of 2 gratings and 2 lenses is known from the literature as a4f system.

A simplified arrangement for the pulse shaper is shown in FIG. 2 b. Inthis case, a mirror S is arranged right after the modulator (2) so thatthe beam runs back into itself with a small vertical offset or at asmall angle. First, this arrangement makes do with few opticalcomponents; second, the light pulses traverse the modulator (2) twice,so that the magnitude of the phase/amplitude modulation is doubled.

FIG. 3 shows schematically the dispersive splitting and combination of ared component r and a blue component b passing the manipulator 2 and thewavelength shape along a direction X to the manipulator 2.

Since the time behavior can be changed in the pulse shaper, the lightpulses pass via corresponding optical components via the microscope Mand the objective V into the specimen P. A nonlinear effect is excitedin the specimen P because of the sharp focussing through the objectiveand the high peak pulse power of the light pulses. This nonlinear effectis recorded by the detector (4). Therefore, a corresponding measurementsignal is available that can be optimized by electronically controllingthe pulse shaper by means of regulation R.

The operation of the regulation will be described by way of example ofgeneration of a two-photon fluorescence signal.

The two-photon fluorescence signal (S) can be described as follows:${S \propto \frac{P_{avg}^{2}}{\tau^{2} \cdot A^{2}}},$where P_(avg) is the average output and T is the pulse length of thelight pulses at the location of the specimen. A stands for the beamcross section at the location of the specimen interaction.

It can be seen from the above equation that the two-photon fluorescencesignal increases as the pulse length and beam cross section decrease andas average output increases. In a microscope, the pulse length isinfluenced, i.e., usually lengthened, by the following factors:

-   -   the glass materials from which the optical elements in the        microscope are made; compensation can be carried out in a        stationary manner;    -   the specimen in itself; in this case, the lengthening of the        pulse depends upon the depth of penetration into the specimen;

further, the pulse widening is generated by higher-order dispersions;therefore, compensation must be carried out for every spectral componentindividually and in real time;

-   -   change in wavelength;    -   change in average output.

The pulse shaper PF, and accordingly the time behavior of the lightpulses, is therefore adjusted by regulation in real time depending onthe above-mentioned variables, wherein the two-photon fluorescencesignal functions as a measured quantity. Essentially the pulse lengthand the average output at the location of specimen interaction areoptimized by the pulse shaper.

Further, the interaction cross sections of the utilized dyes aredependent on the time behavior of the light pulses. Accordingly, it ispossible to optimize the fluorescence signal for individual dyes,wherein the fluorescence of other dyes is simultaneously suppressed.This is known in the literature as coherent control. Thus, by feedingback the measured quantity (in this case, the two-photon fluorescencesignal), it is possible to adjust the time behavior of the light pulsesby phase modulation or amplitude modulation in such a way that thecorresponding measured quantity is optimized.

While the foregoing description and drawings represent the preferredembodiments of the present invention, it will be obvious to one skilledin the art that various changes and modifications may be made thereinwithout departing from the true spirit and scope of the presentinvention.

1. A device for coupling a short pulse laser into a microscope beam pathof a laser scanning microscope used for investigation of nonlinearcontrast methods comprising: a dispersive element for spatiallyseparating the spectral components of the laser radiation; means formanipulating individual spectral components; said manipulating meansacts to manipulate components and the manipulator means is purposefullyoptimized by feeding back a measurement signal and the desiredmeasurement signal is therefore adjusted; and an element for spatiallysuperimposing the manipulated individual spectral components.
 2. Thedevice according to claim 1, wherein, after manipulation, the spectralcomponents are reflected at a mirror and superimposed again by theelement for spatially superimposing.
 3. The device according to claim 1,wherein the microscope is a laser scanning microscope.
 4. The deviceaccording to claim 1, wherein prisms or gratings are used as dispersiveelements.
 5. The device according to claim 1, wherein the manipulatormeans generates an amplitude modulation of the spectral components. 6.The device according to claim 1, wherein the manipulator means generatesa phase modulation of the spectral components.
 7. The device accordingto claim 1, wherein the device is followed by a single-mode fiber forcoupling in a short pulse laser.
 8. The device according to claim 1,wherein a single-mode fiber is also polarization-preserving.
 9. Thedevice according to claim 1, wherein a spatial light modulator is usedin the Fourier plane as a manipulator means.
 10. The device according toclaim 6, wherein the phase modulation in the manipulator means is usedto compensate higher-order dispersion by the use of the feedback. 11.The device according to claim 21, wherein the phase modulation in themanipulator means is optimized depending on the center wavelength of theshort pulse laser by the use of feedback.
 12. The device according toclaim 21, wherein the phase modulation in the manipulator means isoptimized depending on the utilized objective by the use of thefeedback.
 13. The device according to claim 21, wherein the phasemodulation in the manipulator means is optimized depending on theutilized average output by the use of feedback.
 14. The device accordingto claim 21, wherein, by the use of feedback, the phase modulation inthe manipulator means is adjusted depending on the depth of penetrationinto a preparation to be examined and a nonlinearly excited fluorescencesignal is therefore optimized.
 15. The device according to claim 21,wherein a pulse front and a spherical aberration are optimizedadditionally by an adaptive acousto-optic element.
 16. The deviceaccording to claim 21, wherein the phase modulation in the manipulatormeans is optimized depending on the utilized objective by the use offeedback.
 17. The device according to claim 21, wherein a specificexcitation of fluorescence dyes is carried out by phase modulation andamplitude modulation in the manipulator means.
 18. The device accordingto claim 1 wherein the optimization is carried out selectively.
 19. Thedevice according to claim 1, wherein a specific resolution of reactionsin the fluorescence dyes is carried out by phase modulation andamplitude modulation in the manipulator means.
 20. The device accordingto claim 1, wherein a specific bleaching of dyes is carried out by phasemodulation and amplitude modulation in the manipulator means.
 21. Adevice for coupling a short pulse laser into a microscope beam path of alaser scanning microscope comprising: a dispersive element for spatiallyseparating the spectral components of the laser radiation; means formanipulating individual spectral components; wherein the manipulatormeans is purposefully optimized by feeding back a measurement signal andthe desired measurement signal is therefore adjusted; and an element forspatially superimposing the manipulated individual spectral components.22. A laser scanning microscope system comprising: a microscope adispersive element for spatially separating the spectral components of ashort pulse laser that will be coupled into a microscope beam path ofsaid microscope means for manipulating individual spectral components;wherein the manipulating means is purposefully optimized by feeding backa measurement signal and the desired measurement signal is thereforeadjusted; and an element for spatially superimposing the manipulatedindividual spectral components.